28 Cell Death in the Skin

1. INTRODUCTION

During life we are persistently exposed to environmental hazards. A first protective barrier is provided by the skin, protecting us against water loss and external physical, chemical, and biological insults such as wounding, UVB radiation, and microorganisms. The skin consists of an outer squamous epithelium, the epidermis, and an inner connective tissue, the dermis. The barrier is mainly constituted by the epidermis, which is continuously rejuvenated as a result of mitotic activity of the stem cells in the basal layer that provide new keratinocytes. Upon withdrawal from the cell cycle, basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis (Figure 28-1). At the transition from the granular to cornified layer, an increase in intracellular Ca2+ activates transglutaminases, which cross-link different structural proteins beneath the plasma membrane to form the cornified envelope. At the final stage of differentiation, the keratinocytes lose their organelles, including the nucleus, and become the dead, flattened corneocytes. This cell death program, called cornification, has to be well orchestrated because the dead cells act as an essential barrier and fulfill a specific function. Finally, corneocytes are shed from the skin by a process called desquamation. Melanocytes, also residing in the epidermis, are neurectoderm-derived cells that produce melanin, which provides skin pigmentation. Imbalances in the delicate physiologic turnover of proliferating or differentiating keratinocytes can result in the disturbance of the skin barrier function and are reflected in many skin disorders. In addition, improper removal of damaged cells by the terminal differentiation program can result in cancerous lesions.

2. CELL DEATH IN SKIN HOMEOSTASIS

2.1. Cornification and apoptosis

To form cornified envelopes at the outermost epidermal layer, keratinocytes must undergo cell death. Although this process shows parallels with apoptosis, it should be distinguished from it based on morphological and biochemical differences. Apoptosis is a rapid process occurring in single cells that are immediately engulfed by other cells such as macrophages. In vivo, terminal keratinocyte differentiation is a process simultaneously occurring in a whole cell layer, taking up to 2 weeks, and the dead cells fulfill an essential function in the skin barrier before they are shed. It was proposed that apoptosis is blocked during differentiation to prevent premature apoptotic cell death during keratinocyte differentiation and to allow correct cornification. In keratinocytes, the β1-integrins mediate adhesion to the extracellular matrix and regulate the initiation of terminal differentiation and cell-detachment apoptosis, or anoikis. Epidermal keratinocytes differentiate when they detach from the basement membrane and migrate to the suprabasal layers. This differentiation signal is transduced by unoccupied β1-integrins. However, in experimental keratinocyte suspension cultures, unoccupied β1-integrins induce an apoptotic signaling cascade that results in upregulated Bax expression and mitochondrial damage, eventually leading to cell death. This observation implies the existence of an innate survival mechanism required to maintain the viability of suprabasal cells in vivo (Figure 28-2). Activation of the nuclear factor kappa B (NF-κB) transcription factor is one of the antiapoptotic mechanisms that is initiated during keratinocyte differentiation. NF-κB is a complex formed by homoand heterodimerization

Figure 28-2. Antiapoptotic mechanisms during skin homeostasis. During epidermal di erentiation, several prosurvival pathways are activated, including the MAPK, NF-κB, and Akt pathway. The MAPK and Akt pathway can be activated by growth factor receptors, such as epidermal growth factor receptor. How NF-κB is triggered under homeostatic conditions in the skin is currently not clear. It has been suggested that the changed suprabasal keratinocyte interactions mediated by means of adhesion molecules could be involved in NF-κB activation in these layers. MAPK signaling can result in activator protein-1(AP-1) transcription factor activation, which controls proliferation. There exists interplay between Akt and NF-κB because it has been documented that Akt can also activate the NF-κB pathway and NF-κB can induce Akt. However, the molecular details of this interplay are not well known. NF-κB induces the transcription of antiapoptotic genes, whereas Akt can inhibit the proapoptotic protein Bad by phosphorylating it. Phosphorylated Bad can no longer bind and inhibit the prosurvival Bcl-2 family members Bcl-2 and Bcl-XL. Apoptotic signaling can lead to mitochondrial outer membrane permeabilization and cytochrome c release. Cytochrome c will bind (together with (d)ATP) to Apaf-1, resulting in apoptosome assembly and caspase-9 activation. At least in hair follicle cells and melanocytes, it has been shown that Bcl-2 protects these cells against premature apoptosis in homeostatic conditions. Inhibitor of apoptosis proteins (IAPs) can inhibit caspases, thereby hampering the apoptotic capacity of the cell. In addition, IAPs are also transcriptional targets of NF-κB.

which is TNF-R1 and/or JNK dependent. The initiation of skin disease during the first few days after birth may indicate the involvement of environmental factors. These observations imply that NF-κB is of major impor-

tance for the maintenance of a well-balanced immune system in the skin. Although the exact mechanism of inflammation initiation remains unclear, ablation of NF- κB activators from epidermal keratinocytes seems to disturb the immune balance of the skin, resulting in an inflammatory response that is marked by the induction of inflammatory cytokines. Alternatively, mechanisms related to developmental changes of the skin at this early age might be involved in disease initiation. In contrast to IKKβ, IKKα seems to play a crucial role in skin barrier formation, but independent from its NF-κB signaling function. Besides epidermal differentiation, development of hair follicles and limbs and tail also requires IKKα.

Analogous to the skin phenotype observed by ablation of essential NF-κB activating genes, increased NF-κB activation by deletion of IκBα leads to severe skin inflammation, which is TNF, lymphotoxin, and RelA dependent. Tissue-specific deletion of IκBα emphasizes the cross-talk between epidermal and immune cells in the development of skin inflammation. In addition, ablation of RelA from epidermal keratinocytes completely rescued the inflammatory skin phenotype of IκBα-deficient mice. It is noteworthy that in case of over-activation of NF-κB, the inflammatory skin phenotype is T-cell dependent, whereas inactivation of NF-κB results in T-cell independent inflammation. These findings emphasize the important role of NF-κB activation in both keratinocytes and lymphocytes in the development of skin inflammation and underscore the important role of the epidermis as a sensor of inflammatory stimuli.

Other pathways that prevent cell death in the skin are the PI3K/AKT and mitogen-activated protein kinase (MAPK) pathway, both activated by epidermal growth factor receptor. Genetic deletion of Akt1 or Akt1/Akt2 in the mouse results in thinner skin with fewer hair follicles. This phenotype is explained by reduced keratinocyte proliferation. In contrast, knockdown of Akt1, but not of Akt2, in in vitro reconstituted human epidermis results in premature keratinocyte apoptosis and thinner suprabasal layers. Whether the observed difference in Akt implication between the mouse and human epidermis reflects species differences or the fact that in the developing mouse epidermis compensatory effects result in a milder phenotype is currently not clear. Akt can block apoptosis through phosphorylation of the proapoptotic Bcl-2 family member BAD, leading to its inactivation. Importantly, there exists an interplay between the PI3K/Akt and NF-κB pathway, because Akt is believed to activate NF-κB through IKKα and β and NF-κB can induce Akt. The possible link

between Akt and NF-κB was not explored in the Akt1 and Akt2 double-deficient Akt mice.

In both apoptosis and keratinocyte differentiation, protease activity is indispensable. Several skin-specific proteases are required for cornification. The apoptotic caspases, such as caspase-3, -6, -7, and -9, are probably not involved in cornification because they are not activated during differentiation, and deficiency of their genes in the mouse does not result in apparent skin abnormalities. The involvement of caspase- 3 in embryonic skin homeostasis remains the subject of debate, because both the absence and the occurrence of caspase-3 activation has been reported in the developing epidermis in mice. Caspase-14, however, is clearly required for normal skin development. This caspase family member is nonapoptotic, almost exclusively expressed in the suprabasal epidermal cells, and becomes activated during cornification. Caspase-14– deficient mice have defects in proper stratum corneum formation resulting in increased transepidermal water loss and increased sensitivity to UVB irradiation as compared with wild-type mice. Profilaggrin, a direct caspase14 substrate, is not correctly degraded in caspase-14– deficient mice, whereas this is a crucial event in the formation of a completely functional skin barrier. Filaggrin is not only a major CE structural protein, it is also important in skin hydration. In the upper cornified layers of wild-type mice, in contrast to caspase-14–/– mice, filaggrin is further degraded, and this process gives rise to natural moisturizing factors (NMFs) and urocanic acid, a UVB scavenger.

The distinction between apoptotic cell death and cornification is also made at the mitochondrial level. The release of cytochrome c during in vitro keratinocyte terminal differentiation has been reported, but this did not result in apoptosis, although the apoptosome components Apaf-1 and caspase-9 are present in epidermal keratinocytes. Instead, it was suggested that cytochrome c release during keratinocyte differentiation leads to transcription factor activation and gene expression. Antiapoptotic Bcl-2 is downregulated and proapoptotic Bax and Bak are induced in the suprabasal layers of the epidermis, but no abnormalities in skin differentiation have been reported for any of the Bcl-2 family member knockouts. One exception is bcl-2, which is implicated in hair follicle turn-over. Knockout or transgenic mice for this gene show a retardation or acceleration in the first hair follicle growth phase, respectively. In addition, graying occurs during the hair follicle catagen or apoptosisdriven involution phase in Bcl-2-/- mice, probably due to melanocyte apoptosis.

2.2. Death receptors in the skin

The members of the TNF receptor (TNF-R) superfamily are characterized by extracellular cysteine-rich domains that bind their respective ligands and intracellular interaction motifs, such as the death domain (DD) and the TNF-receptor associated factor (TRAF)– binding domain. In general, these receptors are capable of initiating signaling cascades that lead to transcription factor activation and/or cell death. The TNF-R superfamily comprises the so-called death receptors (DRs), namely TNF-R1, Fas, TNF-related apoptosis-inducing ligand (TRAIL) R1 and R2, TRAMP, DR6, EDAR, and p75NTR, which have a cytoplasmic death domain (DD). TNF-R1, Fas, and TRAIL-R and their respective ligands TNF, FasL, and TRAIL are expressed by keratinocytes and have been shown to be able to induce cell death in keratinocytes. In normal conditions, the ligands are poorly expressed or may remain intracellular so that no spontaneous apoptosis occurs, but in pathological conditions (see Section 3, Cell Death in Skin Pathology), activation of these receptors leads to keratinocyte apoptosis.

The nonapoptotic TNF-R family members EDAR, XEDAR, and TROY trigger the ectodysplasin (EDA) pathway, which is of major importance for skin appendage development. The ligands for EDAR and XEDAR (EDAA1 and EDA-A2) are splice variant products of the same gene ED1, whereas TROY is still an orphan receptor. The exact function of the EDA pathway in appendage formation is not known, but mutations in components of the EDA pathway result in hypohidrotic ectodermal dysplasia (HED), manifested by absence or defective formation of hair, teeth, fingers or toes, and sweat glands, which causes overheating. The same phenotype is found in mice that are mutant for EDA or EDAR or its downstream signaling molecules EDAR-associated death domain protein (EDARADD) and TRAF6. Surprisingly, the EDAR death domain receptor does not signal to cell death, but rather to NF-κB to prevent premature cell death during the hair follicle catagen involution phase. X-linked HED (XLHED) can be caused by missense mutations in ED1, resulting in dysfunctional EDA ligand. In mice it was shown that the HED phenotype can be rescued by expression of the correct ligand or prenatal recombinant protein administration of Fc:EDA-A1, a fusion protein of an IgG1 Fc domain with EDA-A1, which facilitates delivery through the placenta and increases the molecule stability. Postnatal injection of Fc:EDA1 in mice and canine XLHED models gave very promising results.